针对含胸腺嘧啶乙二醇DNA的单克隆抗体。

Monoclonal antibody to DNA containing thymine glycol.

作者信息

Leadon S A, Hanawalt P C

出版信息

Mutat Res. 1983 Aug;112(4):191-200. doi: 10.1016/0167-8817(83)90006-8.

Abstract

Exposure of DNA to ionizing or near ultraviolet radiation modifies thymine to form ring-saturated products. One of the major products formed is 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol). Thymine glycol can also be selectively formed by oxidizing DNA with OsO4. We have isolated hybrids that produce monoclonal antibodies against thymine glycol by fusing mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells from BALB/c mice immunized with OsO4-oxidized poly(dT) complexed with methylated bovine serum albumin. This report describes the characterization of the antibody from one hybridoma using a competitive enzyme-linked immunosorbent assay (ELISA). The antibody reacted with both single- and double-stranded DNA treated with OsO4, and with OsO4-treated poly(dA-dT) and poly(dT); it did not crossreact with unmodified or apurinic DNA. It also reacted with DNA treated with H2O2 or with gamma-rays at doses as low as 250 rad. We were able to detect 2 fmoles of thymine glycol in OsO4-treated DNA and could quantitate 1 thymine glycol per 220 000 thymines. Using the antibody and the ELISA, the formation and removal of thymine glycol was examined in cultures of African green monkey cells irradiated with 25 krad of gamma-rays. The antibody reactive sites produced by irradiation (8.5 per 10(6) thymines) were efficiently removed from the cellular DNA.

摘要

将DNA暴露于电离辐射或近紫外辐射会使胸腺嘧啶发生修饰，形成环饱和产物。形成的主要产物之一是5,6 - 二羟基 - 5,6 - 二氢胸腺嘧啶（胸腺嘧啶乙二醇）。胸腺嘧啶乙二醇也可以通过用OsO4氧化DNA来选择性形成。我们通过将小鼠骨髓瘤细胞（P3X63 - Ag8 - 6.5.3）与用与甲基化牛血清白蛋白复合的OsO4氧化的聚（dT）免疫的BALB/c小鼠的脾细胞融合，分离出了产生抗胸腺嘧啶乙二醇单克隆抗体的杂交瘤。本报告描述了使用竞争性酶联免疫吸附测定（ELISA）对一种杂交瘤产生的抗体的特性鉴定。该抗体与经OsO4处理的单链和双链DNA、经OsO4处理的聚（dA - dT）和聚（dT）反应；它与未修饰的或无嘌呤DNA不发生交叉反应。它也与用H2O2或低至250拉德的γ射线处理的DNA反应。我们能够在经OsO4处理的DNA中检测到2飞摩尔的胸腺嘧啶乙二醇，并且能够定量每220000个胸腺嘧啶中有1个胸腺嘧啶乙二醇。使用该抗体和ELISA，在接受25千拉德γ射线照射的非洲绿猴细胞培养物中检测了胸腺嘧啶乙二醇的形成和去除情况。照射产生的抗体反应位点（每10^6个胸腺嘧啶中有8.5个）从细胞DNA中被有效去除。